PROJECT SUMMARY. Protein kinases represent one of the largest gene families and regulate much of biology. Kinase dysfunction is also associated with a plethora of diseases. Although the protein kinase gene families have been mapped onto the kinome, the details of specificity and regulation are buried within each family. These families include not only isoforms but also splice variants for many genes. Splice variants are not represented the kinome, but enormously expand the complexity of signaling networks and specificity. Knockout experiments tell us repeatedly that the isoforms and splice variants are functionally non-redundant, highlighting that the assembly of highly specific complexes within cells and tissues is an essential feature of kinase signaling. Increasingly we are also coming to appreciate the importance of these isoforms and splice variants from disease phenotypes, which further highlights that biology is controlled by finely tuned regulatory networks. cAMP-dependent protein kinase (PKA), expressed in every mammalian cell, regulates fundamental biological processes that include metabolism, development/differentiation, memory, and immune responsiveness. While the PKA C?1 subunit has served in so many ways as the prototypical protein kinase, surprisingly almost nothing is known about the C? isoforms, which include multiple splice variants. While C?1 is ubiquitous in all human cells, expression of the C? isoforms is more tissue-specific, and disease phenotypes suggest that they also are likely to be functionally non-redundant. Our goal here is to characterize three of the C? splice variants that differ only in their first exon. These C? isoforms correlate with several diseases. C?1 leads to cortisol producing adenomas in Cushing?s Disease, ablation of C?2 in immune cells leads to immuno- suppression, and increased C?2 correlates with survival in prostate cancer patients and can cause Carney Complex Disease (CNC) and thyroid tumors. This emphasizes the importance of C? signaling and suggests that our work will have important and previously unappreciated biological and disease relevance. Our innovation lies in the fact that we can easily cross so many scales that extend from basic biochemistry and atomic resolution of the molecules to their isoform-specific distribution in cells and tissues. We will use this multi-scale approach to characterize the structure, function and regulation of three C? isoforms. In parallel we will map the tissue-specific localization of these isoforms in kidney, spleen, thymus and brain using isoform- specific antibodies. Finally we will use a proteomic strategy to identify isoform-specific binding partners. Our broad knowledge of PKA signaling, coupled with our deep understanding of the four functionally non-redundant PKA holoenzymes, provides us with a unique opportunity to explore a wide swath of previously untapped cAMP signaling space. Given the global importance of PKA signaling in all cells, the probability that the C? isoforms will have important physiological as well as disease relevance is high. Our studies will allow us to move forward creatively with developing novel isoform-specific therapies.